Gupta K P, Ward N E, Gravitt K R, Bergman P J, O'Brian C A
Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
J Biol Chem. 1996 Jan 26;271(4):2102-11. doi: 10.1074/jbc.271.4.2102.
The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-alpha-catalyzed P-glycoprotein phosphorylation.
多药耐药（MDR）癌细胞的主要特征是对化疗药物具有广谱抗性，且细胞内药物积累存在明显缺陷，这与药物外排泵P-糖蛋白的过表达相关。蛋白激酶C（PKC）可使P-糖蛋白的连接区磷酸化。已有证据表明，同工酶PKC-α可能与人乳腺癌MCF7-MDR细胞的耐药表型有关，PKC-α在MCF7-MDR细胞中显著过表达，在过表达P-糖蛋白的MCF7构建体中人工过表达PKC-α会显著增强细胞的MDR表型，并伴有P-糖蛋白磷酸化增加。维拉帕米、环孢素A以及其他一些与细胞毒性药物竞争P-糖蛋白结合位点的药物可有效逆转MDR，但这在体内会伴有严重毒性。在本报告中，我们证明一种含有与PKC-α假底物区域相对应序列的N-肉豆蔻酰化肽（P1）通过一种涉及抑制PKC-α的新机制部分逆转了MCF7-MDR细胞中的多药耐药性。P1和另外两种相关的PKC抑制性N-肉豆蔻酰化肽恢复了化疗药物在细胞内的积累，同时抑制了MCF7-MDR细胞中三种PKC-α底物的磷酸化：PKC-α、Raf-1激酶和P-糖蛋白。PKC的第四种N-肉豆蔻酰化肽底物类似物P7不影响MCF7-MDR细胞中的药物积累，也未能抑制PKC-α底物的磷酸化。P1和维拉帕米对MCF7-MDR细胞中药物积累的影响具有相加性。P1不影响P-糖蛋白的表达。MCF7-MDR细胞对P1无交叉耐药性，这表明该肽不是由P-糖蛋白转运的。此外，P1与维拉帕米和环孢素A等MDR逆转剂不同，它不能抑制P-糖蛋白的[3H]叠氮平光亲和标记。实际上，P1增加了MCF7-MDR细胞中P-糖蛋白的[3H]叠氮平光亲和标记，这表明P1对MCF7-MDR细胞中P-糖蛋白的作用不限于抑制泵的磷酸化。P1可能为开发新一代通过涉及抑制PKC-α催化的P-糖蛋白磷酸化的新机制发挥作用的MDR逆转剂提供基础。
